Labeled Chiral Alpha-Hydroxy Ketoacid Derivatives, a Process for Preparing Said Derivatives and Their

(19) TZZ ¥_T (11) EP 2 695 873 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C07B 59/00 (2006.01) C07C 51/367 (2006.01) 26.10.2016 Bulletin 2016/43 C12P 21/02 (2006.01) (21) Application number: 12305984.2 (22) Date of filing: 08.08.2012 (54) Labeled chiral alpha-hydroxy ketoacid derivatives, a process for preparing said derivatives and their use Markierte, chirale alpha-Hydroxy-Ketosäure-Derivate, Verfahren zur Herstellung besagter Derivate und deren Verwendung Dérivés de cétoacides alpha-hydroxylés chiraux étiquetés, procédé de préparation de ces dérivés et leur utilisation (84) Designated Contracting States: WO-A1-2011/083356 US-A1- 2006 148 042 AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO • DAVID H.H. CROUT ET AL.: "The Base-catalysed PL PT RO RS SE SI SK SM TR Rearrangement of a-Acetolactate (2-Hydroxy-2-methyl-3-oxobutanoate): a Novel (43) Date of publication of application: Carboxalte Ion Migration in a Tertiary Ketol 12.02.2014 Bulletin 2014/07 Rearrangement", J. CHEM. SOC. PERKIN I, 1979, pages 1982-1989, (73) Proprietors: • D.J. ROBINS ET AL.: "Pyrrolizidine alkaloids. The • COMMISSARIAT A L’ENERGIE ATOMIQUE ET absolute configuration of C-2 in Mono-crotalic AUX acid", J. CHEM. SOC. (C), 1970, pages 1334-1336, ENERGIES ALTERNATIVES • RICHARD K. HILL ET AL.: "Stereochemistry of 75015 Paris (FR) Valineand Isoleucine Biosynthesis IV. Synthesis, • Centre National de la Recherche Scientifique Configuration and Enzymatic Specificity of 75016 Paris (FR) alpha-Acetolactate and alpha-Aceto-alpha-hydroxybutyrate", (72) Inventors: BIOORGANIC CHEMISTRY, vol. 8, 1979, pages • Plevin, Michael 175-179, 38000 GRENOBLE (FR) • FRANK B. ARMSTRONG ET AL.: "Biosynthesis •Kerfah,Rime of Valine and Isoleucine: Synthesis and 38600 FONTAINE (FR) Biological Activity of (2S)-alpha-Acetolactic Acid • Pessey, Ombeline (2-Hydroxy-2-methyl-3-oxobutanoic Acid), and 38600 FONTAINE (FR) (2R)- and (2S)-alpha-Acetohydroxybutyric Acid • Boisbouvier, Jérome (2-Ethyl-2-hydroxy-3-oxobutanoic Acid)", J. 38660 Saint Vincent de Mercuze (FR) CHEM. SOC. PERKIN I, 1983, pages 1197-1201, • Gans, Pierre • J. CHEM. SOC. PERKIN I, vol. 1979, 1979, pages 38340 VOREPPE (FR) 1982-1989, • J. CHEM. SOC. (C), vol. 1970, 1970, pages (74) Representative: Gevers & Orès 1334-1336, 41 avenue de Friedland • BIOORG. CHEM., vol. 8, 1979, pages 175-189, 75008 Paris (FR) • J. CHEM. SOC. PERKIN TRANS I, vol. 1983, 1983, pages 1197-1201, (56) References cited: Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 695 873 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 695 873 B1 Description [0001] The present invention relates to labeled chiral alpha-hydroxy ketoacid derivatives, a process for preparing said derivatives and their use for isotopic labeling of amino acids, in particular, for isotopic labeling of methyl groups of amino 5 acids, and more particularly, for specific isotopic labeling of valine, leucine and isoleucine methyl groups, in proteins and biomolecular assemblies. [0002] The invention also concerns a process for analyzing proteins and biomolecular assemblies by NMR spectroscopy comprising a step of isotopic labeling of amino acids, in particular, valine, leucine and isoleucine, in proteins and biomolecular assemblies to be analyzed by the chiral alpha-hydroxy ketoacid derivatives of the invention. 10 [0003] The invention further relates to a kit for isotopic labeling of valine, leucine and isoleucine amino acids, in proteins and biomolecular assemblies, comprising one or more chiral alpha-hydroxy ketoacid derivatives of the invention. [0004] The use of solution NMR spectroscopy to analyze the dynamics, interactions and function of large proteins (> 100 kDa) and supra-molecular systems is becoming increasingly feasible. Key to this advance has been the development of new and powerful ways of labeling biomolecules has been critical for stimulating many of the advances in NMR 15 methodology. [0005] Methyl groups have been proven to be ideal molecular probes for solution NMR spectroscopy studies of large proteins. [0006] In initial methyl-labeling procedures, alpha-keto acids were used as precursors in the production of methyl- protonated isoleucine (Ile). A more recent strategy is based on the use of labelled 2-(S)-2-hydroxy-2-ethyl-3-oxobu- 20 tanoate, a biosynthetic precursor of isoleucine in E. Coli (Ayala I. et al., Chem Comm., 2011, www.rsc.org/chemcomm, DOI: 10.1039/C1CC12932E). In this method, the precursor is chemically synthetized meaning that the precursor is obtained as a racemic mixture and that only half of the mixture obtained can be converted by the bacteria. [0007] Valine (Val), leucine (Leu) and isoleucine (Ile) are three amino acids of great interest as their methyl groups account for more than 50% of all methyl probes available in proteins. 25 [0008] Protonation of leucine and valine methyl groups in perdeuterated proteins is commonly achieved using methyl protonated 2-oxo-3-methylbutanoic acid (also known as alpha-ketoisovalerate), an intermediate in the biosynthesis of these amino acids, in which both methyl groups are 1H, 13C-labeled. The use of this type of alpha-ketoisovalerate proved inefficient in high-molecular-weight proteins as it results in overcrowded 1[H,13C]-correlated spectra due to the sheer number of NMR-visible methyl probes. 30 [0009] Ruschak A.M. et al., J Biomol NMR, 2010, 48(3), p.129-35 and Ayala I. et al., J. Chem Commun, 2012, 48, p. 1434-1436 describe synthetic routes for preparing ester derivatives of 2-hydroxy-2-methyl-3-oxobutanoic acid and 2- hydroxy-2-ethyl-3-oxobutanoate for the specific labeling of Isoleucine gamma-2 methyl groups. In these references, the compounds are prepared by chemical synthesis from methyl (or ethyl) acetoacetate and are thus obtained in the form of a racemic mixture. Consequently, only half of the compound obtained - with the S stereochemistry - can be incorporated 35 by the bacteria. Moreover, the derivatives of 2-hydroxy-2-methyl-3-oxobutanoic acid and 2-hydroxy-2-ethyl-3-oxobutanoate are in the form of an ester, meaning that an additional step is required to deprotect the compounds in basic medium before use. Under poorly controlled reaction conditions, this may result in a significant degradation of the compounds. [0010] Godoy-Ruiz R. et al., J. Am. Chem. Soc., 2010, 132(51), p.18340-50 describe simultaneous selective isotope 40 labeling of Alanine, Leucine, Valine and Isoleucine methyl positions using alpha-ketoisovalerate (for labeling Ala, Leu and Val sites) and alpha-ketobutyrate (for labeling of Ile positions) and their use to obtain distance restraints and mobility data. The labeling method described in this reference leads to isotopic leaks in the gamma-2 position of Isoleucines resulting in artifacts in the extraction of structural constraints. This phenomenon is due to the deamination of some of the labeled 3-13C-alanine leading to the in vivo synthesis of labeled pyruvate. The condensation of the thus obtained 45 labeled pyruvate with endogenous 2-oxobutanoate in the presence of the acetolactate synthase, yields 2-hydroxy-2- ethyl-3-oxo-4-13C-butanoate. [0011] Engel, S. et al., Biotechnology & Bioengineering, 88, p.825-83, reports the use of acetohydroxyacid synthase I (AHAS I) from Escherichia Coli in the stereoselective synthesis of aromatic alpha-hydroxy ketones. [0012] US 2006/0148042 relates to a biotransformation process for the preparation of chiral aromatic alpha-hydroxy 50 ketones using acetohydroxyacid synthase (AHAS) or tartronate semialdehyde synthase (TSAS). This document is totally silent regarding the synthesis of non aromatic synthesis of chiral acetohydroxy acids, the synthesis of chiral acetohydroxy acids specificallyor entirelydeuterated and/or enriched incarbon 13 ( 13C) and their use for isotopic labeling of amino acids. [0013] Thus, there remains a need for labeled stereospecific alpha-hydroxyl ketoacids that are capable of efficiently and specifically label amino acids, in particular the methyl groups of amino acids selected from leucine, valine and 55 isoleucine. [0014] In particular, there remains a need for labeled stereospecific alpha-hydroxy ketoacids as described above, that are manufactured by a regioselective and stereospecific process in high yields and under mild conditions. [0015] More particularly, there remains a need for labeled stereospecific alpha-hydroxy ketoacids as described above, 2 EP 2 695 873 B1 capable of labeling the amino acid without causing isotopic leaks at the site where the amino acid is labeled, i.e. the methyl group of amino acids leucine, valine and isoleucine. [0016] Even more particularly, there remains a need for labeled stereospecific alpha-hydroxy ketoacids as described above, that can be incorporated into the target protein by the bacteria without detectable scrambling. 5 [0017] The present invention addresses these and other needs in the art by providing a compound of formula (I) having an (S) configuration: 10 15 wherein - X1 and X2 are, independently from each other, 1H (H) or 2H (D); - Y1, Y2 and Y3 are, independently from each other, 12C (C) or 13C; 20 - R1 is a methyl group in which the carbon atom is 12C (C) or 11C and the hydrogen atoms are, independently from each other, 1H (H) or 2H (D); - R2 is a methyl group in which the carbon atom is 12C (C) or 13C and the hydrogen atoms are, independently from each other, 1H (H) or 2H (D); or - R2 is an ethyl group in which the carbon atoms are, independently from each other, 12C (C) or 13C and the hydrogen 25 atoms are, independently from each other, 1H (H) or 2H (D), with the proviso that in compound of formula (I), at the same time, at least one hydrogen atom is 2H (D) and at least one carbon atom is 13C.

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